Spectrophotometers are useful in many analyte detection regimes as they can continuously and simultaneously measure the result of light-matter interaction at multiple wavelengths. The interaction usually takes the form of absorbance as light is transmitted through the sample, but may also apply to changes as the light is scattered from the sample, or emission of light as a result of light incident upon the sample. Of these interactions, light absorbance is a particularly useful method. The wavelengths at which an analyte absorbs light is indicative of the type of analyte, and the amount of light absorbed at these wavelengths is proportional to that analyte's concentration. These features of absorbance are intrinsic to the analyte and are theoretically independent of the measurement instrument. It is these features that can allow for the application of sophisticated mathematical analysis techniques to establish accurate determination of analyte concentrations over a wide range of states or conditions of the analyte. These capabilities are described in the literature, for example see Baylor and O'Rourke (L. C. Baylor and P. E. O'Rourke, “UV-Vis for On-Line Analysis”, in “Process Analytical Technology”, K. A. Bakeev, Ed., Blackwell Publishing, Oxford, U K, 2005, Ch. 6).
Unfortunately real instruments distort measurement of intrinsic absorbance features by what is commonly known as the instrument response function. In some situations this function can be determined and can be used to correct measurements back to theoretical values. However the function is not constant but can drift over time. The multi-wavelength capability of spectrophotometers can allow for real-time diagnostics that can better correct for drift of the instrument response function.
Another advantage of the spectrophotometer is that by measuring absorbance at multiple wavelengths, a great deal of information can be acquired that can be used to distinguish various sources of signal changes. These capabilities allow the spectrophotometer to be used in situations where the properties of the sample may be changing over time, e.g. as a monitor for a particular analyte in a chemical process. As a result of such capabilities, for certain systems the accuracy of analyte detection and measurement with spectrophotometers can be higher than with other detection approaches.
Elements of spectrophotometer design such as hardware, control software, and data analysis technique are chosen to maximize these advantages and minimize instrument response effects. For example, spectrophotometers for absorbance or reflectance measurements generally include two spectrometers. One spectrometer is utilized as a reference spectrometer and is dedicated to monitoring the incident light intensity. The other spectrometer measures the light intensity after interaction with the sample. The sample spectrometer readings can be corrected for variations in the incident light intensity by dividing them by the reference spectrometer readings. Thus, overall intensity drift is corrected and changes in instrument output can be better correlated to changes in the sample.
Unfortunately, there are also difficulties in utilizing spectrophotometers. For instance, in many applications, there is no capability to calibrate/validate instrument performance in-line with a process operation. As such, there may be no way to continually monitor and confirm the instrument performance and calibration without uninstalling the spectrometers, or at least interrupting process operations in order to perform instrument checks.
Accurate calibration and validation of a spectrophotometer is not merely desirable from the standpoint of optimizing performance, however. It is critical for the instrument to work properly. Calculating sample absorbance requires comparison of the intensities of the same wavelengths from the two spectrometers and even though the spectrometers may have identical designs, their wavelength-to-pixel calibrations will not be identical and are subject to wavelength drift typically associated with temperature changes and component aging. Over time the wavelength calibration of the two spectrometers drift apart. Therefore, in order to function accurately, there must be a way to convert the wavelength-to-pixel calibrations for the sample and reference spectrometers to a common basis for accurate comparison. Similarly, corrections must be made to assure that the response to intensity changes is proportionally identical for both spectrometers.
What are needed in the art are spectrophotometers that can more accurately detect analytes and measure their concentrations. More specifically, spectrophotometers that can be accurately calibrated for each spectral acquisition and continuously correct for wavelength and intensity drift so as to provide spectral data of an “ideal virtual instrument”, free of instrument response effects, would be of great benefit. Moreover, the ability to provide such on-going calibration and continuous correction while maintaining a spectrophotometer in-line would be highly beneficial.